DESCRIPTION (Verbatim from the Applicant's Abstract): These studies address the mechanisms by which G protein-coupled receptors (GPCR) achieve their discrete localization in target cells. Localization is determined by targeting mechanisms that govern receptor delivery to the surface and tethering mechanisms that govern receptor stability at its final surface "destination." Studies funded by this grant, focusing on alpha2AR subtypes and the Al adenosine receptor as models of GPCR, have revealed that targeting to the apical (A1AdoR) or basolateral (alpha2AR subtypes) surface is driven by multiple, independent and hierarchical sequences in or near the bilayer. In contrast, tethering to the basolateral surface involves the large third intracellular (3i) loop of alpha2AR subtypes. We identified two interacting proteins for the alpha2AR subtype 3i loops: 14-3-3 proteins and spinophilin, both multi-domain proteins implicated as scaffolds in various signaling pathways. The aims of this proposal are to 1) determine if spinophilin or 14-3-3 proteins tether the alpha2AR subtypes to the basolateral surface of MDCK II cells, a model system for polarized renal epithelia, 2) determine the trafficking itinerary of wildtype V2 vasopressin receptors in MDCK II cells and the point(s) at which varying alleles of the V2R that contribute to X-Iinked nephrogenic diabetes insipidus (NDI) are interrupted in their direct delivery to the basolateral surface, and the cell surface "rescue" of these mutant V2R by temperature shifts or by chemical chaperones, and 3) use molecular cloning strategies to identify cDNAs encoding molecules that rescue cell surface expression of mutant V2R. We will also explore whether these cDNAs can enrich the cell surface expression of the alpha2cAR subtype, which exists predominantly in an intracellular precursor pool in a variety of cell backgrounds. There are two reasons for including another GPCR, i.e. the V2R, as a model system in our studies to understand the mechanisms underlying GPCR localization. First, our finding that multiple, non-contiguous membrane-embedded sequences dictate the delivery of GPCR to polarized surfaces in MDCKII cells means that a single sequence region cannot serve as a "ligand" to identify the targeting machinery for these receptors. Second, many diseases result from intracellular accumulation of molecules that must achieve surface expression to regulate cell function; cDNAs that rescue cell surface expression of intracellularly trapped receptors identified in the proposed studies likely encode targets for therapeutic intervention not only in NDI, but also in retinitis pigmentosa, cystic fibrosis, and other inherited diseases.